The invention refers to the use of antagonists against LA-paf and novel very high affinity receptors on endothelial cells. According to the invention, endothelial cells express very high affinity receptors for paf, LA-paf and/or lipoproteins during their differentiation. Specific antagonists against very high affinity receptors, by preference on endothelial cells are tested using novel screening procedures to identify agents which may be clinically useful against endothelial cell-mediated diseases. According to the invention very high affinity receptors, by preference on endothelial cells, are involved with the transport of LA-paf and/or lipoproteins. LA-paf antagonists and antagonists against very high affinity receptors are used to treat or prevent recruitment of lipids. The undesirable side effects of lipid recruitment, pathological differentiation, irritation, aging or death of cells are thereby avoided. Antagonists can be administered by preference with food or beauty products to individuals requiring said substances. The invention successfully tests antagonists against LA-paf or very high affinity receptors on endothelial cells using novel methods.
The invention is based on novel methods based on several discoveries of the inventor. Very high affinity receptors on freshly prepared endothelial cells are compared here with receptors produced during differentiation of endothelial cell lines in response to growth factors such as insulin. The novel very high affinity receptors interfere with the inflow of lipid mediators to be enriched in the subendothelium. Thus, antagonists provide protection against fluid escape. Lowering of the endothelium barrier by pathological differentiation leads to cell emigration and/or edema formation with increased thickness of the basal lamina. Bacterial, cerebral, genetic, metabolic, inflammatory and allergic diseases can result from lowering the endothelial cell barrier with further adherence and/or emigration of blood cells to be prevented by antagonists against very high affinity receptors on endothelial cells.
Moreover, the inventor found out that LA-paf and lipoproteins constitute vehicles to transport lipids through the endothelium to the subendothelium mediated by an intermediate of very high affinity receptors. Cells such as platelets, monocytes or endothelial cells produce and release LA-paf which is associated with lipoproteins in the plasma. LA-paf is produced only in cells and is related to phospholipid metabolism, for example, during peroxidation. Thus, very high affinity receptors interfere either with lipoprotein transport through the endothelial monolayer or with emigration of activated blood cells such as monocytes which produce LA-paf and carry LA-paf from inside the vessels out.
LA-paf is an ether phospholipid which is physicochemically and functionally distinct as compared with chemically defined paf. The priority document of the present invention discloses at least two binding sites for LA-paf, with one binding site being accessible only for lipoproteins (normal or modified) and LA-paf U.S. patent application Ser. No. 08/104,599!.
LA-paf is a preformed mediator present in healthy persons. According to the invention, LA-paf is a novel risk factor in metabolic and genetic diseases. This theory is based on the following findings: 1) Cells involved in atherosclerosis such as platelets, endothelial cells or monocyte/macrophage like cells synthesize LA-paf; 2) the atherogenetic low density lipoprotein (LDL) and cholesterol cause expression of specific paf binding sites on monocyte/macrophage like cells carrying LA-paf from inside the vessels out 3) lipoproteins contain LA-paf with a common binding site, for example, on platelets and 4) very high affinity receptors on endothelial cells interfere with the lipoprotein transport from inside the vessels out. This is shown here and in the priority documents R. Korth, U.S. patent application Ser. No. 07/994,752 and European Patent, Publication No. 0312 913 B1 with the priority document P 37 35 525.2, Germany!.
Based on these discoveries, the invention discloses novel methods to find antagonists against LA-paf or very high affinity receptors. Endothelial cells express "very high affinity receptors", as compared with tenfold lower affinity "high affinity paf receptors" on blood cells, cancer cells or cell lines. Endothelial cells and blood cells express distinct receptors on their surface which are antagonized by distinct and specific antagonists. Immortalized endothelial cell lines, for example, express very high affinity receptors during their differentiation. Antagonists against various receptors for paf, lyso paf, lipoproteins or LA-paf can be tested on the surface or inside of cells.
Based on these findings antagonists are administered to persons with indicated risk factors to maintain the integrity of both the endothelium and the subendothelium and to prevent and treat diseases as mentioned. Very high affinity receptors on endothelial cells are blocked by antagonists to inhibit the binding of LA-paf, paf, blood cells and/or lipoproteins and to reduce the transport of pathological compounds, for instance lipids, through the endothelium. Antagonists against very high affinity receptors by preference on endothelial cells are not defined prior to the present invention. Simple methods select antagonists against LA-paf receptors, paf metabolism and LA-paf production using preferably platelets, neutrophils, eosinophils, lymphocytes, monocytes, tissue cells including adipocytes, or cell lines. The methods and their use are fully described here and in the priority documents of the present invention U.S. patent application Ser. No. 08/104,599, European Patent Application, Publication No. 0 459 432 A1!.
Antagonists
Substances that antagonize very high affinity receptors or LA-paf can be selected from the group consisting of triazolothieno-diazepines or analogs, as well as homologous compounds. In addition ginkgolides and paf analogues, such as CV 3988, are suitable. Triazolothieno-diazepines and ginkgolides are paf antagonists as described R. Korth (1989) Brit. J. Pharmacol. 98, 653-661; R. Korth (1988) Eur. J. Pharmacol. 152, 101-110!. Of the triazolothieno-diazepine compounds WEB 2086 and WEB 2098 are especially suitable. Of the ginkgolides BN 52020, BN 52021 and a mixture of BN 52020, BN 52021 and BN 52022, which is referred to as BN 52063, achieve the best results. The synthetic compound BN 50739 T. Yue et al., J. Exp. Pharmacol. Exp. Therapeutics 257, 1991, pp 374-381! is used here as an example for synthetic ginkgolides because it contains the active chemical structures of both ginkgolides and hetrazepines. The chemical term of CV 3988 is rac-3-(N-n-octadecyl carbamoyl oxy)-2-methoxypropyl 2-tiazolioethyl phosphate; the term of WEB 2086 is 3-(4-(2-chlorophenyl)-9-methyl-6H-thieno(3,2-f) (1,2,4)triazolo-(4,3-a)-(1,4) diazepine-2yl)-1-(4-morpholinyl)-1-propanone; the term of WEB 2098 is (3-(4-(2-chlorophenyl)-9-cyclopropyl-6H-thieno(3,2-f)-(1,2,4)triazolo-(4,3 -a)(1,4)diazepine-2yl)-1-(4-morpholinyl)-1-propanone; the term of BN 52020 is 9H-1, 7a-Epoxymethano)-1H, 6aH-cyclopenta(c)furo(2,3-b)furo(3',2':3,4) cyclopenta(1,2-d)furan-5,9,12(4H)-trione, 3-tert-butylhexahydro-4, 7b-dihydroxy-8-methyl; the term of BN 52021 is 9H-1, 7a-Epoxymethano)-1H,6aH-cyclopenta(c)furo(2,3-b)furo-(3',2':3,4) cyclopenta(1,2-d)furan-5,9,12(4H)-trione, 3 tert-butylhexahydro-4, 4b-11-trihydroxy-8-methyl; and the term of BN 52022 is 9H-1, 7a-(Epoxymethano)-1H, 6aH-cyclo-penta(c)furo(3',2':3,4)cyclopenta (1,2-d)furan-5,9,12(4H)-trione, 3 tert-butyl hexahydro-2,4,7b,11-tetrahydroxy-8-methyl. The chemical term of BN 50739 is tetrahydro-4,7,8,10methyl(chloro-2 phenyl)6(dimethoxy-3,4-phenyl)thio) methylthiocarbonyl-9pyrido(4',3'-4, 5)thieno(3,2-f)triazolo-1,2,4(4,3-a)diazepine-1,4).
The antagonists can be administered topically, orally, parenterally, by liposomes, food, beauty products, syrups or inhalation. According to the invention paf or LA-paf antagonists can also be administered in form of food for example with "FIDA infants' foods for invalids and seniors" R. Korth U.S. Trademark Application, Ser. No. 74/416,579!. The compounds are administered as active ingredients in conventional pharmaceutical preparations, e.g. in compositions comprising an inert pharmaceutical vehicle and an effective dose of the active substance, such as tablets, coated tablets, capsules, lozenges, powders, solutions, suspensions, aerosols for inhalation, ointments, emulsions, syrups, food, suppositories, etc.
An emulsion has been prepared containing ginkgolides for topical use adding a commercially available ginkgolide solution (Tebonin forte, Schwabe, Karlsruhe, Germany) to commercially available "pH-5 Eucerin Intensivlotio" (Beiersdorf, Hamburg, Germany), for example 30 gtt ad 125.0 ml. Ginkgolides can also be added to food. Triazolothieno-diazepines, preferably lipophilic compounds, can be used as well. An effective dosage of the agents according to the invention is preferably between 0.01 and 50 mg. For oral administration, between 3 and 20 mg/kg are used. For intravenous or intramuscular administration, between 0.01 and 50 mg, or between 0.1 and 10 mg/kg are used. For inhalation, solutions should be used containing, for example, 0.01 to 1.0% of the agent.
BN 50739 has been administered to endotoxin-treated rabbits (3 and 10 mg/kg i.p., IHG, Le Plessis Robinson, France in Yue et al., J. Pharmacology and Experimental Therapeutics 254, May 1990, pp. 976-981). Stransky et al. describe novel Thieno-1,4-Diazepines (U.S. Pat. No. 4,900,729) with doses up to 200 mg/kg p.o. using guinea pigs.
According to the invention natural Ginkgo biloba is administered for the first time by food for example "FIDA-infants' foods for invalids and seniors" against irritation, pathological differentiation, irritation, aging, degeneration and death of cells in vivo. The administration of ginkgolides, preferably with food, is suitable for example for older persons and children. The invention is suitable to compose and produce FIDA-infants' foods for invalids and seniors.
Beauty products can be tested and produced for their clinical use to create new vessels and to prevent and treat aging, edema formation as well as immunological, genetic or metabolic disorders. Beauty products are administered, for example, topically using pharmaceutical vehicles by preference with acid pH values.
Clinical use
A clinical relevance of the invention is shown because LA-paf can be considered as a novel risk factor. Freshly prepared endothelial cells express very high affinity receptors and LA-paf is present in human cells and lipoproteins. According to the invention, specific antagonists against LA-paf and very high affinity receptors can be used for protection of the endothelial barrier in vivo. They are specifically useful to enhance by preference the creation of new vessels and/or to treat and prevent pathological water osmoregulation with irritation of the tissue, for example, the lymphatic system, neuronal cells, the brain, the skin, the kidney, and the heart prematurely growing old. They protect against an increased thickness of the basal lamina and/or the subendothelial tissue. This protects, for example, in cases of diabetes, against blindness or kidney diseases. The cause of diabetes cannot yet be treated, but persons with risk factors such as hyperglycemia, obesity, hyperinsulinemia, acid pH values and/or hyperlipidemia can be treated as long as they are healthy, to protect their tissue, particularly their vascular system, their eyes, their heart and their kidneys. Antagonists prevent edema formation, pathological irritation, differentiation, aging and death of cells. Pathological differentiation of endothelial cells, caused preferably by insulin, cholesterol, lipoproteins (normal or modified) or acid pH values has a considerable importance in metabolic, addictive, inflammatory, allergic, vascular, chronicle, idiopathic, genetic diseases and transplantation. Protection of healthy endothelial cells by creation of new vessels can be mediated by administration of compounds of the invention, by preference with food and beauty products.
Antagonists with an effect on the central nervous system protect either the endothelial blood brain barrier via an intermediate of cell adherence, or emigration and/or neuronal cells. Endothelial cells produce here lyso paf, and lyso paf is a novel neurotransmitter interacting with specific lyso paf receptors on neutrophils and/or neuronal cells in the priority document of the present invention R. Korth, U.S. patent application Ser. No. 08/261,765!. Antagonists against chronic endothelial cell irritation with production of lyso paf and/or activation of the protein kinase C protect, for example, against neoplastic, mental, neuronal or inflammatory diseases.
Water intoxication has been shown to be a serious problem in many patients with chronic disorders. For example, seniors, diabetics or psychiatric patients have polydipsia and hyponatremia with unexplained defects in urinary dilution, osmoregulation of water intake and vasopressin secretion M. B. Goldman et al. (1990) New Engl. J. Med. 318, pp 397-403!. Medicaments containing bilobalid are used to treat nervous diseases I. Anadere et al. (1985) Clinical Hemorheology 5, pp 411-420!, but ginkgolides protect here, for the first time, healthy endothelial blood brain barrier to prevent and antagonize episodic defects in osmoregulation.
The increase of prostacyclin in response to antagonists against endothelial very high affinity receptors is a novelty and proposes a novel clinical use. Indicated antagonists are clinically used to protect the vascular system and/or to trigger the synthesis and release of prostacyclin, for example, during administration of prostaglandin antagonists such as aspirin.
The tumor promoting factor PMA which has considerable importance in cancer causes expression of paf receptors on non-differentiated cells R. Korth et al., Chem. Phys. Lipids 70 (1994) 109-119!. Thus, the selected compounds might also protect against cancer disease or metastasis.
Leukocytes such as neutrophils, eosinophils, monocytes or lymphocytes are involved in various inflammatory reactions such as allergy, cell adherence and migration, as well as bacterial or fungal phagocytosis. Novel antagonists inhibit .sup.3 H!paf binding and cellular functions such as calcium flux. Novel binding assays demonstrate the differences between paf receptors on the surface of intact cells. For example, intact human platelets, blood neutrophils and eosinophils have different paf, LA-paf and lyso paf receptors determined with binding assays using unlabelled paf, LA-paf or lyso paf (500 nM) with or without specific paf, lyso paf and LA-paf antagonist. These antagonists are by preference ginkgolides or triazolothieno-diazepines. A K.sub.d value of 0.44 nM with 3612 high affinity (ha) receptors per neutrophil, a K.sub.d value of 9.2 nM with 4100 lyso paf receptors per neutrophil and a K.sub.d value of 5.5 nM with 8.6.times.10.sup.4 moderate affinity (ma) receptors per eosinophil are calculated using Scatchard plot analysis of the saturated specific binding. LDL, cholesterol and Lapaf significantly upregulated these receptors on platelets with paf and lyso paf upregulation of neutrophils and low dose paf (not lyso paf) downregulation of eosinophil receptors. Intact neutrophils, monocytes and endothelial cells, but not platelets and eosinophils metabolize added paf and all these cells produce and carry LA-paf. These experiments are fully described in the priority documents (U.S. patent application Ser. Nos. 08/104,599; 08/261,765; 08/246,476 and 08/172,234).
According to the invention, novel methods are used to select specific antagonists against high, very high or moderate affinity sites for paf, lipoproteins, LA-paf and/or lyso paf on various cells. Clinically the selected compounds are used for the prevention and treatment of genetic diseases by preference of risk factors such as obesity, hyperlipidemia with or without arteriosclerosis, for neuronal, cerebral, mental, psychotic diseases. The novel compounds are administered to treat and prevent vascular and skin diseases with oedema formation or metabolic disorders. They reduce side effects of drugs such as glucocorticosteroids, cAMP modulating drugs, antihistamines and prostaglandin antagonists. Leukocyte-, lipoprotein-, and/or endothelial cell-mediated diseases are involved in degenerative, allergic, cancer and inflammatory disorders to be treated with compounds selected as described (U.S. patent application Ser. Nos. 08/104,599; 08/261,765; 08/246,476 and 08/172,234).
Novel Methods
The present invention discloses novel methods to test the efficacy of antagonists using, for example, endothelial cell lines. Differentiated cells are compared with non-differentiated endothelial cell lines such as immortalized endothelial cells, for example, umbilical vein endothelial cells (Im-Huvec). This invention also discloses quick and simple methods of endothelium differentiation with various factors such as growth factors, such as insulin.
According to the invention, endothelial cells express very high affinity (vha) receptors during differentiation as compared with nearly no receptors on non-differentiated cells, such as Im-Huvec cells see Example 1. Freshly prepared endothelial cells express in vivo, very high affinity receptors as shown in Example 2. According to the invention very high affinity receptors interfere with the binding and metabolism of paf and LA-paf in endothelial cells, platelets and monocyte/macrophage like cells. See Examples 3 and 4 and the priority document U.S. patent application Ser. No. 08/104,599!. Thus, antagonists are selected with novel methods which are fully described in the examples to successfully inhibit very high affinity receptors, LA-paf binding, LA-paf effects and/or production.
According to the invention, gene transfer of differentiated endothelial cells into soluble cells is proposed using a standard procedure described for high affinity paf receptors from guinea pig lungs and blood cells Z. Honda et al. (1991) Nature 349, 342-346!. The invention discloses a procedure to test antagonists of very high affinity paf receptors on soluble cells or endothelial cells cultured, for example, on soluble particles.
In the case that endothelial cells are used, the procedure of the present invention can be used to determine the activity of specific paf or LA-paf antagonists. According to the invention, endothelial cells, for example, freshly prepared or immortalized endothelial cells after differentiation, are used to test the efficacy and specificity of antagonists. In order to conduct a quick and simple test on the efficacy with regard to their antagonistic activity vis-a-vis paf or LA-paf receptors, i.e. to use a screening procedure, for example, to find effective antagonists which can then be taken into consideration for treating or preventing disorders caused by pathological irritation, differentiation, aging or death of cells, then according to the invention, the best method is to proceed as follows.
a) Endothelial cells are cultured or non-differentiated endothelial cells are mixed in culture with at least one compound of various differentiation factors, for example, lipids, (lipo)proteins, LA-paf, hormones such as steroids, for example, cholesterol, growth factors such as insulin or erythropoietin etc., inflammatory mediators such as LA-paf or cytokines, compounds such as PMA or DMSO and acid medium, PA1 b) a given quantity of purified cells is mixed with a given quantity of labelled paf or LA-paf and the antagonist to be determined, PA1 c) a given quantity of the same purified cells is mixed with a given quantity of labelled paf or LA-paf in the absence of antagonists, PA1 d) the cells are separated from the mixtures b) and c) in each case, PA1 e) the quantity of labelled paf or LA-paf bound to the cells is measured in each case, and PA1 f) the efficacy of the paf or LA-paf antagonist is determined from the relationship between the quantity of labelled paf or LA-paf which is bound to the cells according to b) in the presence of the antagonist on one hand, and the quantity of labelled paf or LA-paf which is bound to the cells according to c) in the absence of the antagonist on the other hand. PA1 a) Endothelial cells are cultured, PA1 b) endothelial cells are mixed with at least one differentiation factor producing endothelial receptors for example the growth factor insulin, PA1 c) endothelial cells are preferably washed with acid pH values and binding studies with paf, LA-paf, analogues of paf, antagonists or antibodies are performed as described above and according to step b) and c) treated endothelial cells are compared with non-treated cells in the presence and absence of labelled and unlabelled agonists, antibodies and antagonists, PA1 d) genes producing endothelial receptors for paf or LA-paf according to b) and c) are transferred into soluble cells for example cell lines, cancer cells, fungal cells or bacterial cells using standard procedures or endothelial cells are cultured on soluble particles, PA1 e) soluble cells and/or soluble particles with endothelial cells expressing very high affinity receptors are used to test substances, for example, specifically related against high, very high or moderate affinity receptors for paf, LA-paf with analogues or lipoproteins using by preference screening procedures with automatons. PA1 a) cell lines are cultured in the presence of serum; PA1 b) labelled or unlabelled paf or analogues are added; PA1 c) cells are extracted using standard procedures for phospholipid extraction.
Preferably human umbilical vein endothelial cells (Huvec) or non-differentiated endothelial cells, for example, immortalized cells (Im-Huvec) after differentiation, are used for the procedure. It is also suitable to use venous or arterial endothelial cells from human or animal tissues. The method of the invention determines the efficacy of LA-paf antagonists or antagonists specifically directed against very high affinity receptors on endothelial cells. Other cells such as platelets, neutrophils, eosinophils, lymphocytes, monocytes, tissue cells including adipocytes and cell lines such as monocyte-macrophage like cells, are also used to test antagonists or to produce LA-paf.
Differentiation of cell lines, for example, of Im-Huvec can be performed by non-specific cell stimulation. Cells can be treated, for example, with growth factors, lipids, steroids hormones and inflammatory mediators such as cholesterol, LDL, LA-paf, insulin, or cytokines. Infection, activation of the protein kinase C, and acid medium cause expression of the indicated receptors. Cells can be treated during a shorter incubation period with acid pH values and PMA, for example, to activate the protein kinase C. Cell lines such as U 937 cells express paf receptors during bacterial or fungal infection. The invention is suitable to select specific antagonists (and antibodies) for a clinical use against the pathogenesis of metabolic, genetic, inflammatory and/or neoplastic disorders.
The ligand of binding studies according to step c) d) e) f) can be paf or LA-paf, their analogues, and antagonists with analogues or antibodies in a marked, fluorescent or colored form. As labelled ligands agonists, antagonists or antibodies can be used with and without labelled and unlabelled, colored or fluorescence labelled paf, LA-paf with analogues, antibodies and antagonists.
The mixing according to the steps b) and c) is done preferably at a temperature of 20.degree. C. or 4.degree. C. After mixing, the cells are incubated for 30 min to several hours, or, overnight, before they are separated according to step d) of the procedure of the present invention. Endothelial cells are preferably washed with acid buffer and detached after the binding procedure in a cool medium with EDTA and without Magnesium and Calcium. It is known that the cells are preferably separated from the buffer by centrifugation or filtration. The separation of the cells according to step d) can be performed by filtration or centrifugation. After this, the quantity of labelled paf or LA-paf which is (specifically) bound to the cells is determined. If radioactively labelled paf or LA-paf are used, only the radioactivity bound to cells is measured. The radioactivity bound in the filter without cells is subtracted from these values.
By drawing calibration graphs, which are obtained with varying quantities of the antagonist in accordance with step b), it is thus possible to obtain the efficacy of the antagonist at a 50% inhibitory value, i.e. as that quantity of the antagonist which, in relation to a given quantity of cells, leads to a 50% inhibition of the reversible binding of the labelled ligand. It is also possible to calculate binding kinetics of antagonists.
Antibodies, by preference monoclonal antibodies, against LA-paf itself or paf receptors can be formed for diagnostic tests or the prevention and treatment of diseases. According to the invention antibodies can be tested or produced using soluble cells, for example, antibodies with agonist and antagonist potency using radio- and fluorescence labelled or colored compounds. The difference between differentiated vs. non-differentiated cells with presence and absence of paf or LA-paf receptors can be used to produce antibodies using standard procedures. Antibodies against surface proteins of non-differentiated cells are subtracted from those on differentiated cells using screening procedures according to the invention. The invention refers also to production of LA-paf in large amounts which is an essential condition for production of antibodies or of enzyme assays. Antibodies, for example, against lipoproteins are used successfully in the priority document U.S. patent application Ser. No. 08/104,599, FIG. 5!.
Diagnostic tests for the clinical use can be facilitated using special testing containers which are commercial available and which are modified for smaller blood volumes as proposed in German utility model application G 87 16 004.8.
Since the specific binding of paf, LA-paf and analogues is in close correlation with the cellular signal transduction and cellular calcium stream, the procedure of the present invention can also be used for measuring paf, LA-paf and agonists, for example, by comparing their effects on the cellular calcium stream with calibration graphs of synthetic paf or mediator production and release as described R. Korth (1989) Brit. J. Pharmacol. 98, 653-661!.
By preference endothelial cells cultured on dishes or on soluble particles but also soluble cells, for example, cell lines, fungal cells, cancer cells or bacterial cells are used, by preference after gene transfer. These cells express distinct receptors for paf, LA-paf, lipoproteins, lyso paf and paf analogues to select distinct antagonists.
In order to perform a simple procedure to test antagonists preferably against very high affinity receptors on endothelial cells genes can be enriched, defined and transferred, for example, into soluble cells, by preference cell lines and bacterial cells. The invention refers to transfer, by preference genes interfering with very high affinity receptors or LA-paf production to soluble cells. Next, screening procedures can be performed preferably by automatons. The antagonists can be tested according to the following procedure:
The culture of endothelial cells, for example, Huvec or endothelial cell lines, for example, Im-Huvec can be performed preferably with standard culture techniques. The endothelial cells, for example immortalized endothelial cells, can be cultured also on soluble particles to further facilitate the procedure. The culture dishes and soluble particles can also be mixed for example with gelatin and growth factors, hormones, mediators, lipids and antagonists. Endothelial cells can also be grown on smooth muscle cells to define and treat cellular interactions leading to vascular regulation. In this context, functional assays can be used, for example, cell adherence, calcium influx or production and release of mediators in the absence and presence of antagonists and antibodies. According to the invention binding studies are performed with unlabelled and/or labelled agonists, antagonists, antibodies, hormones, lipids or (lipo)proteins as described above. The expression of genes for paf or LA-paf receptors, for example, in response to insulin the binding studies according to b) and c) can be defined and enriched, for example, during culture (2 to 24 h) in the presence of 2 I.E. human insulin. Other growth factors and compounds can be added to a standard medium. Compounds including pharmacological substances can be added to antagonize the production and expression of genes encoding paf or LA-paf receptors as well as LA-paf production.
It is suitable to use soluble cells instead of adherent cells to facilitate screening procedures of new compounds according to e). The compounds are, for example, antibodies and antagonists directed by preference against very high affinity paf receptors for LA-paf, lipoproteins or paf. Alternatively endothelial cells can be cultured on soluble particles. The latter possibility is also suitable because various cellular transduction mechanisms might define the structure of novel receptor protein(s). Genes might produce very high affinity receptors, paf and/or LA-paf receptor(s) by preference with different cell-specific structures but also phosphorylation and/or modulating proteins etc. might be expressed during differentiation.
Antagonists inhibit either receptor-dependent interactions and metabolism of paf and LA-paf or LA-paf production. According to the invention, LA-paf is produced during oxidation of long chain fatty acid derivatives such as the cellular paf metabolite alkyl-acyl-glyceryl-phosphorylcholine. Oxidation of long chain fatty acid paf metabolites triggers LA-paf production to be used or to compose, for example, food containing antioxidant substances such as vitamins. Lipoproteins are used here for the first time as growth factors to differentiate cells with expression of paf receptors. This interferes with the cellular transport of lipoproteins and/or LA-paf from inside the vessels out. Lipoproteins bind directly to very high affinity receptors on the endothelium with or without intermediate of peroxidation.
The invention refers to in vitro production of LA-paf by cell lines. Cellular LA-paf production is a quick and simple method to produce large quantities of labelled and unlabelled LA-paf, for example, to perform binding studies with labelled and/or unlabelled LA-paf or to produce antibodies as described above. The invention is also suitable to test and produce food and beauty products interfering, by preference with peroxidation and LA-paf production. When cell lines are used to produce labelled or unlabelled LA-paf then according to the invention, the best method is to proceed as follows:
According to a) the cell lines are by preference monocytemacrophage like cells. Blood and tissue cells, cancer cells bacterial as well as fungal cells can be used in vivo and in vitro using all culture techniques.
According to b) lipids, oxidized phospholipids, ether phospholipids, labelled or unlabelled paf or LA-paf with analogues can be added which are metabolized by cell lines. Resident blood cells as well as monocyte/macrophage like cells produce and carry unlabelled LA-paf without addition of paf.
According to c) all procedures of phospholipid extraction and/or purification can be used by preference HPLC with a biological assay to quantify and purify labelled or unlabelled LA-paf.